AU2009222998B2

AU2009222998B2 - Anti-TYRP1 antibodies

Info

Publication number: AU2009222998B2
Application number: AU2009222998A
Authority: AU
Inventors: Paul J. Balderes; Xiaoqiang Kang
Original assignee: ImClone LLC
Current assignee: ImClone LLC
Priority date: 2008-03-12
Filing date: 2009-03-11
Publication date: 2013-05-23
Anticipated expiration: 2029-03-11
Also published as: CL2009000567A1; KR20100113631A; UA99339C2; CN101970500A; TWI384997B; KR101203777B1; AR070821A1; NZ587305A; EA019517B1; EP2268672A1; AU2009222998A1; IL207581A0; MX2010010021A; US7951370B2; CN101970500B; US20090232823A1; JP2011516041A; CA2718289A1; EA201071068A1; BRPI0909633A2

Abstract

The present invention provides for fully human antibodies and chimeric antibodies that bind human TYRP1 antigen with an affinity comparable to or higher than TA99, a murine antibody specific to TYRP1. The invention further provides polynucleic acids and host cells that encode and express these antibodies. The invention also provides for methods of modulating activity of TYRP1, treating growth of a cancer cell, and treating a malignant melanoma in mammals by administering an effective amount of an antibody either alone or in combination with an anti-cancer agent or treatment.

Description

WO 2009/114585 PCT/US2009/036739 -1 ANTI-TYRP1 ANTIBODIES CROSS-REFERENCE This application claims the benefit of U.S. Provisional Application No. 61/069199 5 filed March 12, 2008. FIELD OF THE INVENTION The present invention is directed to human and chimeric antibodies, including fragments or portions thereof, that are specific to human tyrosinase-related protein-1 10 (TYRPi). The antibodies are used for treating growth of cancer cells and can be used alone or in combination with an anti-neoplastic agent or treatment. BACKGROUND OF THE INVENTION Human tyrosinase-related protein-1 (TYRP1), also known as gp75, (WO 15 91/14775) (SEQ ID NO: 28) is a melanosomal membrane glycoprotein involved in melanin biosynthesis. It is found predominantly within the melanosomes of melanocytes and is also found expressed on the cell surface of melanocytes and human melanomas. The TYRP1 antigen is highly immunogenic. Antibodies and T-cells to TYRP1 have been identified in melanoma patients. It appears that both cellular and humoral 20 responses are effective in eliminating melanoma in vivo. Adoptive transfer of melanoma reactive T-cells also results in tumor regression. Antibody response induced by TYRPI vaccine also could inhibit melanoma growth and metastasis in animals. Although a variety treatment options for melanoma have been studied including, small molecule inhibitors, chemotherapeutics, immunotherapies including vaccines (for 25 example United States Patent 6,168,946), gene therapy/immunostimulants, and anti angiogenics, at the present time, there are no effective therapies for patients with melanoma. Development of new treatments for this unmet medical need is highly warranted. Animal studies have resulted in the discovery of the antibody TA99. TA99 30 (IgG2a), a murine monoclonal antibody (MAb) specific for human and murine TYRP 1, localizes to subcutaneous human melanoma in vivo. See Welt et al., Proc. NatL. Acad.

WO 2009/114585 PCT/US2009/036739 -2 Sci. USA 84: 4200-4204 (1987) and United States Patent 4,798,790. TA99 treatment inhibited tumor growth and metastasis in mice. See Takechi et al., Clin Cancer Res. 2:1837-42 (1996). Mice treated with TA99 often lose hair color (depigmentation), suggesting 5 destruction of melanocyte in the skin. The Fc receptor-mediated effector activation appears to play a critical role in the elimination of cells targeted by TA99. The anti tumor effect of TA99 was dramatically reduced in FcR knockout mice. See Clynes et al., Proc. Nati. Acad. Sci. USA 95:652-656 (1998). However, the murine nature of TA99 means it would be precluded from use as a therapeutic in humans due to potential issues 10 of immunogenicity and further its ability to activate downstream immune effector functions would be limited. Thus there is a need to provide alternative anti-TYRP 1 antibodies which are effective in the treatment of melanoma. The present invention provides alternative anti TYRP 1 antibodies that are effective in the treatment of melanoma. 15 Additionally, there is a need to provide alternative anti-TYRP 1 antibodies which have improved binding affinity for TYRP 1 compared with those antibodies known in the art. The present invention provides alternative anti-TYRP 1 antibodies which have improved binding affinity for TYRP 1 compared with those antibodies known in the art. Further, there is a need to provide alternative anti-TYRPI antibodies which have 20 reduced immunogenicity in humans and improved ability to activate downstream immune effector functions such as antibody dependent cellular cytotoxicity (ADCC). The present invention provides chimeric and human anti-TYRPI antibodies which have reduced immunogenicity in humans and an improved ability to activate downstream immune effector functions such as antibody dependent cellular cytotoxicity (ADCC) compared 25 with those antibodies known in the art. There also remains a need to provide alternative anti-TYRPI antibodies which have improved stability via a reduction in protein misfolding and incorrect processing. Preferred antibodies of the present invention have improved stability via reduced protein misfolding and incorrect processing. 30 SUMMARY OF THE INVENTION 3 The disclosure herein is directed to human and chimeric monoclonal antibodies, and fragments thereof, that bind to the melanoma antigen TYRPI (SEQ ID NO:28). According to a first aspect of the present invention, there is provided a monoclonal antibody or fragment thereof that specifically binds human TYRP 1 (SEQ ID NO: 28), comprising a VH s comprising the sequence QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLESMGWINTNTGN PTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRYSSSWYLDYWGQGTLV TVSS (SEQ ID NO: 14), a CDRL1 having the sequence RASQSVSSYLA (SEQ ID NO:4), a CDRL2 having the sequence DASNRAT (SEQ ID NO:5), and a CDRL3 having the sequence io QQRSNWLMYT (SEQ ID NO:6). According to a second aspect of the present invention, there is provided an isolated polynucleic acid comprising a nucleotide sequence encoding the antibody or fragment in accordance with the first aspect of the present invention. According to a third aspect of the present invention, there is provided an expression vector 15 comprising the polynucleic acid in accordance with the second aspect of the present invention operably linked to expression control elements such that the encoded antibody or fragment may be expressed. According to a fourth aspect of the present invention, there is provided a recombinant cell comprising the expression vector in accordance with the third aspect of the present invention, 20 which recombinant cell is capable of producing an antibody or fragment in accordance with the first aspect of the present invention. According to a fifth aspect of the present invention, there is provided an antibody or fragment produced by culturing the recombinant cell in accordance with the fourth aspect of the present invention so that the antibody or fragment is produced and recovering the antibody or fragment 25 from the culture.

3a According to a sixth aspect of the present invention, there is provided an antibody or fragment in accordance with the first or fifth aspect of the present invention for use as a medicament. According to a seventh aspect of the present invention, there is provided the use of an antibody or fragment thereof in accordance with the first or fifth aspect of the present invention for the 5 manufacture of a medicament for treating or preventing a condition in a subject indicating administration of said antibody or fragment thereof. According to an eighth aspect of the present invention, there is provided the use of an antibody or fragment thereof in accordance with the first or fifth aspect of the present invention for the manufacture of a medicament for treating cancer in a subject. to According to a ninth aspect of the present invention, there is provided a method for treating or preventing a condition in a subject indicating administration of a TYRP1-binding antibody or fragment thereof, said method comprising administering to said subject an antibody or fragment thereof in accordance with the first or fifth aspect of the present invention. According to a tenth aspect of the present invention, there is provided a method for treating a is cancer in a subject, said method comprising administering to said subject an antibody or fragment thereof in accordance with the first or fifth aspect of the present invention.

3b One embodiment of the present invention is a monoclonal antibody that specifically binds human TYRPI with a dissociation constant, KD, at ambient laboratory 5 temperature (20 *C - 25 *C), in the range from 0.1 x 10~9 M to 1.6 x 10~9 M, wherein said KD values are determined by surface plasmon resonance. In other embodiments of the present invention, the monoclonal antibody is chimeric or human. Fragments of such antibodies, which fragments retain the ability to specifically bind human TYRP1, are part of the invention, even though dissociation constants for such fragments are not in the 10 specified range. In other embodiments of the present invention, the monoclonal antibody specifically binds human TYRP1 with a KD of about 0.1 x 10-9 M to about 1.2 x 10-9 M, about 0.1 x 10~ 9 M to about 0.8 x 10-9 M, about 0.1 x 10-9 M to about 0.4 x 10-9 M, about 0.2 x 10-9 M to about 1.2 x 10-9 M, about 0.2 x 10-9 M to about 0.8 x 10-9 M, about 0.2 x 15 10- 9 M to about 0.4 x 10~9 M, about 0.2 x 10~ 9 M to about 0.3 x 10-9 M, or about 0.28 x 10-9 M. One embodiment of the present invention is an antibody or fragment thereof that binds TYRPI comprising a CDRH I having the sequence GYTFTSYAMN (SEQ ID NO:1), a CDRH2 having the sequence WINTNTGNPTYAQGFTG (SEQ ID NO:2), a 20 CDRH3 having the sequence RYSSSWYLDY (SEQ ID NO:3), a CDRL1 having the sequence RASQSVSSYLA (SEQ ID NO:4), a CDRL2 having the sequence DASNRAT (SEQ ID NO:5), and a CDRL3 having the sequence QQRSNWLMYT (SEQ ID NO:6), wherein said antibody further comprises an amino acid substitution within one of said CDR sequences. In another embodiment, the aforementioned CDRs do not have an 25 amino acid substitution in one of the CDR sequences. In yet another embodiment, the antibody having the aforementioned CDRs specifically binds human TYRP1 with a dissociation constant KD in the range from 0.1 x 10~9 M to 1.6 x 10-9 M. In another embodiment of the present invention, the antibody or fragment thereof that specifically binds TYRP 1, comprising a CDRH 1 having the sequence WO 2009/114585 PCT/US2009/036739 -4 GFNIKDYFLH (SEQ ID NO:7), a CDRH2 having the sequence WINPDNGNTVYDPKFQG (SEQ ID NO:8), a CDRH3 having the sequence DYTYEKAALDY (SEQ ID NO:9), a CDRL1 having the sequence RASGNIYNYLA (SEQ ID NO:10), a CDRL2 having the sequence DAKTLAD (SEQ ID NO:1 1), and a 5 CDRL3 having the sequence QHFWSLPFT (SEQ ID NO: 12), further comprises an amino acid substitution within one of said CDR sequences. In another embodiment, the aforementioned CDRs (SEQ ID NOs:7-12) do not have any amino acid substitutions. Another embodiment of the present invention is an antibody or fragment thereof that binds TYRP1, and comprises 10 a VL having the sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDA SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWLMYTFGQG TKLEIK (SEQ ID NO: 16) and a VH sequence of 15 QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLECMG WINTNTGNPTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRY SSSWYLDYWGQGTLVTVSS (SEQ ID NO: 13) or QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLESMG 20 WINTNTGNPTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRY SSSWYLDYWGQGTLVTVSS (SEQ ID NO: 14). Another embodiment of the present invention is a monoclonal antibody comprising a heavy chain of SEQ ID NO: 29 and a light chain of SEQ ID NO: 32; or a heavy chain of SEQ ID NO: 30 and a light chain of SEQ ID NO: 32. In one embodiment, 25 an antibody comprises two heavy chains of SEQ ID NO: 29 and two light chains of SEQ ID NO: 32, or comprises two heavy chains of SEQ ID NO: 30 and two light chains of SEQ ID NO: 32. TYRP 1-binding fragments of such antibodies are part of the invention. The present invention is also directed to isolated DNAs encoding such antibodies and portions thereof. Other embodiments of the present invention include: an isolated 30 polynucleic acid comprising a nucleotide sequence encoding the antibody, or a fragment thereof; an expression vector comprising said nucleotide sequence linked to an expression WO 2009/114585 PCT/US2009/036739 -5 sequence or a recombinant host cell comprising said expression vector or a recombinant host cell or a progeny thereof, wherein said cell expresses the antibody, or fragment thereof Yet another embodiment of the present invention is a method of producing or purifying an antibody, or fragment thereof, comprising culturing said cells under 5 conditions permitting expression of the antibody or fragment thereof. Additionally, the present invention is directed to methods of inhibiting growth of a cancer cell, and methods of treating melanoma, all in mammals, by administering an effective amount of antibody. One embodiment of the present invention is using the previously described antibodies or fragments thereof as a medicament. In yet another 10 embodiment, the previously described antibodies or fragments thereof are to be used in the treatment of cancer, including but not limited to malignant melanoma. In another embodiment, the invention provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of cancer. In a preferred embodiment the cancer is malignant melanoma. 15 The antibodies may be used alone or in combination with an anti-neoplastic agent or treatment. One embodiment of the present invention is using the previously described antibodies in combination with an additional anti-cancer agent or treatment. In yet another embodiment, the anti-cancer agent is dacarbazine. 20 DETAILED DESCRIPTION OF THE INVENTION The present invention provides for human and chimeric antibodies, and fragments thereof, specific to the TYRPI antigen, as well as isolated or purified polynucleotide sequences encoding the antibodies. Antibodies of the present invention can be used to treat neoplastic diseases, including solid and non-solid tumors, and for treatment of 25 hyperproliferative disorders. The term antibody includes fragments that bind TYRP-1, unless otherwise noted. Binding parameters herein are for full-length antibodies, not fragments, which will necessarily have different binding parameters because of their different size. Naturally occurring antibodies typically have two identical heavy chains and two 30 identical light chains with each light chain covalently linked to a heavy chain by an inter chain disulfide bond. Multiple disulfide bonds further link the two heavy chains to one WO 2009/114585 PCT/US2009/036739 -6 another. As used herein, the term "antibody" includes immunoglobulin molecules comprising 4 polypeptide chains, two heavy (H) chains and two light (L) chains inter connected by disulfide bonds. Individual chains can fold into domains having similar sizes (110-125 amino acids) and structures, but different functions. 5 The light chain can comprise one variable domain (abbreviated herein as VL) and/or one constant domain (abbreviated herein as CL). The light chains of antibodies (immunoglobulins) are either kappa (K) light chains or lambda (k) light chains. The expression VL, as used herein, is intended to include both the variable regions from kappa-type light chains (VK) and from lambda-type light chains (V). The light chain 10 constant region is comprised of one domain, CL. The heavy chain can also comprise one variable domain (abbreviated herein as VH) and/or, depending on the class or isotype of antibody, three or four constant domains (CHI, CH2, CH3 and CH4) (abbreviated herein collectively as CH). In humans, the isotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further subdivided into 15 subclasses or subtypes (IgA 1

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2 and IgG 1 -4). The present invention includes antibodies of any of the aforementioned classes or subclasses (isotypes). Human IgGI is the preferred isotype for the antibodies of the present invention. Generally, the variable domains show considerable amino acid sequence variability from one antibody to the next, particularly at the location of the antigen 20 binding site. Three regions, called hypervariable or complementarity-determining regions (CDRs), are found in each of VL and VH, which are supported by less variable regions called framework regions (FR). Amino acids are assigned to a particular CDR region or domain in accordance with Kabat convention (Kabat, et al., Ann. NY Acad. Sci. 190:382 93 (1971); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, 25 U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3 CDR3-FR4. The portion of an antibody consisting of VL and VH domains is designated Fv 30 (Fragment variable) and constitutes the antigen-binding site. Single chain Fv (scFv) is an antibody fragment containing a VL domain and a VH domain on one polypeptide chain, WO 2009/114585 PCT/US2009/036739 -7 wherein the N terminus of one domain and the C terminus of the other domain are joined by a flexible linker (see, e.g., U.S. Pat. No. 4,946,778 (Ladner et al.); WO 88/09344, (Huston et al.); WO 92/01047 (McCafferty et al.) describes the display of scFv fragments on the surface of soluble recombinant genetic display packages, such as bacteriophage. 5 The peptide linkers used to produce the single chain antibodies can be flexible peptides selected to assure that the proper three-dimensional folding of the VL and VH domains occurs. The linker is generally 10 to 50 amino acid residues. Preferably, the linker is 10 to 30 amino acid residues. More preferably the linker is 12 to 30 amino acid residues. The most preferable linker is 15 to 25 amino acid residues. An example of such 10 linker peptides includes (Gly-Gly-Gly-Gly-Ser)3. An "isolated antibody" is an antibody that (1) has been partially, substantially, or fully purified from a mixture of components; (2) has been identified and separated and/or recovered from a component of its natural environment; (3) is monoclonal; (4) is free of other proteins from the same species; (5) is expressed by a cell from a different species; 15 or (6) does not occur in nature. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Examples of isolated antibodies include an antibody that has been affinity purified, an antibody that has been made by a hybridoma or other cell line in vitro, and a human 20 antibody derived from a transgenic mouse. The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are substantially identical except for possible naturally occurring mutations or minor post-translational variations that may be present. 25 Monoclonal antibodies are highly specific, being directed against a single antigenic site (also known as determinant or epitope). Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the 30 antibody as being obtained from a substantially homogeneous population of antibodies, WO 2009/114585 PCT/US2009/036739 -8 and is not to be construed as requiring production of the antibody by any particular method. The term "antibodies," as used herein, also includes "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or 5 homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., mouse or rat) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological 10 activity. See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Thus, the present invention includes, for example, chimeric antibodies comprising a chimeric heavy chain and/or a chimeric light chain. The chimeric heavy chain may comprise any of the heavy chain variable (VH) regions described herein or mutants or variants thereof fused to a heavy chain constant region of a non-human antibody. The 15 chimeric light chain may comprise any of the light chain variable (VL) regions described herein or mutants or variants thereof fused to a light chain constant region of a non human antibody. The term "human antibody," as used herein, includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences (as 20 described in Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the 25 CDRs. The human antibody can have at least one position replaced with an amino acid residue, e.g., an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human 30 framework sequences.

WO 2009/114585 PCT/US2009/036739 -9 The phrase "recombinant human antibody" includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated 5 from an animal that is transgenic for human immunoglobulin genes, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (See, Kabat, et al., supra.). 10 Fc (Fragment crystallization) is the designation for the portion or fragment of an antibody that comprises paired heavy chain constant domains. In an IgG antibody, for example, the Fc comprises CH2 and CH3 domains. The Fc of an IgA or an IgM antibody further comprises a CH4 domain. The Fc is associated with Fc receptor binding, activation of complement-mediated cytotoxicity and ADCC. For antibodies such as IgA 15 and IgM, which are complexes of multiple IgG like proteins, complex formation requires Fc constant domains. Thus, antibodies of the invention include, but are not limited to, naturally occurring antibodies, antibodies, human antibodies, humanized antibodies, recombinant human antibodies, monoclonal antibodies, digestion fragments, specified portions and 20 variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof; each containing at least one CDR. Functional fragments include antigen binding fragments that bind to a TYRP1 antigen. For example, antibody fragments capable of binding to TYRP 1 or a portion thereof, and which are embraced by the present invention 25 include bivalent fragments such as (Fab') 2 with inter-chain disulfide bonds intact, monovalent fragments such as Fab (Fragment, antigen binding) which refers to the fragments of the antibody consisting of VL CL VL CH1 domains and do not retain the heavy chain hinge region (e.g., by papain digestion), fabs which retain the heavy chain hinge region, facb (e.g., by plasmin digestion), F(ab') 2 , Fab' which lack disulfide bonds, 30 pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and re-aggregation) and Fv or scFv (e.g., by molecular biology techniques). Antibody WO 2009/114585 PCT/US2009/036739 -10 fragments are also intended to include, e.g., domain deleted antibodies, linear antibodies, single chain antibodies, scFv, single domain antibodies, multivalent single chain antibodies, multi-specific antibodies formed from antibody fragments including diabodies, triabodies, and the like that bind specifically with antigens. 5 The hinge region separates the Fab and Fc portions of the antibody, providing for mobility of Fabs relative to each other and relative to Fc, as well as including multiple disulfide bonds for covalent linkage of the two heavy chains. The antibodies, or fragments thereof, of the present invention are specific for TYRPl. Antibody specificity refers to selective recognition of the antibody for a 10 particular epitope of an antigen. Antibodies, or fragments thereof, of the present invention, for example, can be mono-specific, bi-specific, or multi-specific. Bi-specific antibodies (BsAbs) are antibodies that have two different antigen-binding specificities or sites. Multi-specific antibodies have more than two different antigen-binding specificities or sites. Where an antibody has more than one specificity, the recognized epitopes can be 15 associated with a single antigen or with more than one antigen. Thus, the present invention provides bi-specific antibodies, or fragments thereof, that bind to two different antigens, with at least one specificity for TYRP 1. The present invention provides isolated antibodies or fragments thereof specific for TYRP 1. The TYRP 1 protein is mammalian, and is preferably human. In an especially 20 preferred embodiment, an antibody of the invention is capable of binding to both human TYRPI and murine TYRPI [Shibahara et al., Nucleic Acids Res. 14(6) 2413-2427 (1986)] and is hence useful in both both pre-clinical and clinical in vivo studies. The antibodies of the invention are capable of one or more of the following activities: 1) displaying high affinity binding towards TYRP1 exclusively; and 2) inhibiting tumor 25 growth in vitro and in vivo. Specificity of the present antibodies, or fragments thereof, for TYRPI can be determined based on affinity and/or avidity. Affinity, represented by the equilibrium constant for the dissociation of an antigen with an antibody (KD), measures the binding strength between an antigenic determinant and an antibody-binding site. 30 Antibodies of the present invention, or fragments thereof, also include those for which binding characteristics have been modified or improved by direct mutation, WO 2009/114585 PCT/US2009/036739 -11 methods of affinity maturation, phage display, or chain shuffling. Affinity and specificity can be modified or improved by mutating CDRs and screening for antigen binding sites having the desired characteristics (see, e.g., Yang et al., J. Mol. Biol., (1995) 254: 392 403). CDRs are mutated in a variety of ways. One way is to randomize individual 5 residues or combinations of residues so that in a population of otherwise identical antigen binding sites, all twenty amino acids are found at particular positions. Alternatively, mutations are induced over a range of CDR residues by error prone PCR methods (see, e.g., Hawkins et al., J. Mol. Biol., (1992) 226: 889-896). For example, phage display vectors containing heavy and light chain variable region genes can be propagated in 10 mutator strains of . coli (see, e.g., Low et al., J. Mol. Biol., (1996) 250: 359-368). These methods of mutagenesis are illustrative of the many methods known to one of skill in the art. A convenient way for generating substitutional variants is affinity maturation using phage display. Briefly, several CDR region sites are mutated to generate all 15 possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity, specificity, IC50, EC50, KD) as herein disclosed. In order to identify candidate CDR region sites for modification, 20 alanine scanning mutagenesis can be performed to identify CDR region residues contributing significantly to antigen binding. Alternatively, or in addition, random mutagenesis may be performed on one or more CDR sequences at one or more residue positions, either while the CDR is operably linked to the variable region or while the CDR is independent of other variable region sequence and then the altered CDR returned 25 to a variable region using recombinant DNA technology. Once such variant antibodies are generated and expressed, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. As described herein, in addition to the antibodies specifically described herein, 30 other "substantially homologous" modified antibodies can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled WO 2009/114585 PCT/US2009/036739 -12 in the art. For example, the framework regions can vary from the native sequences at the primary structure level by several amino acid substitutions, terminal and intermediate additions and deletions, and the like. Moreover, a variety of different human framework regions may be used singly or in combination as a basis for the humanized 5 immunoglobulins of the present invention. In general, modifications of the genes may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis. The present invention includes TYRP1-binding polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or 10 hypervariable regions of the described full-length anti-TYRPI antibodies. Substantially the same amino acid sequence is defined herein as a sequence with at least 70%, preferably at least about 80%, and more preferably at least about 90%, 95%, 96%, 97%, 98%, or 99% homology to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman (Proc. Natl. Acad. Sci. USA 15 (1988) 85:2444-8). The antibodies of the present invention, or fragments thereof, include human antibodies having one, two, three, four, five, and/or six complementarity determining regions (CDRs) selected from the group consisting of the amino acid sequences of the CDRs as set forth in Table 1. 20 In another embodiment, the present antibodies, or fragments thereof, can have a heavy chain variable region of 20D7 or 20D7S and/or a light chain variable region of 20D7 or 20D7S, outlined below. 20D7 and 20D7S are particularly preferred antibodies of the present invention. These antibodies have human VH and VL framework regions (FRs) as well as human CDRs. 25 The present invention includes nucleic acid sequences that encode an anti-TYRPI antibody heavy chain, comprising any one of the VH regions or a portion thereof, or any one of the VH CDRs, including any variants thereof, as disclosed herein. The invention also includes nucleic acid molecules that encode an anti-TYRP1 antibody light chain comprising any one of the VL regions or a portion thereof or any one of the VL CDRs, 30 including any variants thereof as disclosed herein.

WO 2009/114585 PCT/US2009/036739 -13 Each domain of the antibodies of this invention can be a complete antibody with the heavy or light chain variable domain, or it can be a functional equivalent or a mutant or derivative of a naturally-occurring domain, or a synthetic domain constructed, for example, in vitro using a technique such as one described in WO 93/11236 (Griffiths et 5 al.). For instance, it is possible to join together domains corresponding to antibody variable domains, which are missing at least one amino acid. Also included is an antibody with one or more amino acid substitution, mutation or deletion within one of the CDR sequences. The important characterizing feature is the ability of each domain to associate with a complementary domain to form an antigen-binding site. Accordingly, 10 the terms variable heavy and light chain fragment should not be construed to exclude variants, including variants to the CDRs, that do not have a material effect on specificity. The antibodies of the present invention may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein, Nature 256: 495-497 (1975) and Campbell, Monoclonal Antibody Technology, 15 The Production and Characterization of Rodent and Human Hybridomas, Burdon et al., Eds., Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well as by the recombinant DNA method described by Huse et al., Science 246: 1275-1281 (1989). Antibody fragments can be produced by cleaving a whole antibody, or by 20 expressing DNA that encodes the fragment. Fragments of antibodies may be prepared by methods described by Lamoyi et al., J. Immunol. Methods 56: 235-243 (1983) and by Parham, J. Immunol. 131: 2895-2902 (1983). Such fragments may contain one or both Fab fragments or the F(ab') 2 fragment. Such fragments may also contain single-chain fragment variable region antibodies, i.e. scFv, diabodies, or other antibody fragments. 25 Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. 239,400; PCT Application WO 89/09622; European Patent Application 338,745; and European Patent Application EP 332,424. Preferred host cells for transformation of vectors and expression of the antibodies of the present invention are mammalian cells, e.g., NSO cells (non-secreting (0) mouse 30 myeloma cells), 293 and CHO cells and other cell lines of lymphoid origin such as WO 2009/114585 PCT/US2009/036739 -14 lymphoma, myeloma, or hybridoma cells. Other eukaryotic hosts, such as yeasts, can be alternatively used. The transformed host cells are cultured by methods known in the art in a liquid medium containing assimilable sources of carbon (carbohydrates such as glucose or 5 lactose), nitrogen (amino acids, peptides, proteins or their degradation products such as peptones, ammonium salts or the like), and inorganic salts (sulfates, phosphates and/or carbonates of sodium, potassium, magnesium and calcium). The medium furthermore contains, for example, growth-promoting substances, such as trace elements, for example iron, zinc, manganese and the like. 10 Where it is desired to express a gene construct in yeast, a suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7. Stinchcomb et al. Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of 15 the trp 1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene. The antibodies of the invention may be isolated or purified by any method known 20 in the art, including precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immuno-affinity chromatography as well as gel filtration or zone electrophoresis. A preferred method of purification for the antibodies of the current invention is Protein-A affinity chromatography. 25 DNA encoding human antibodies can be prepared by recombining DNA encoding human constant regions and variable regions, other than the CDRs, derived substantially or exclusively from the corresponding human antibody regions and DNA encoding CDRs derived from a human. Suitable sources of DNAs that encode fragments of antibodies include any cell, 30 such as hybridomas and spleen cells, which express the full-length antibody. The fragments may be used by themselves as antibody equivalents, or may be recombined into WO 2009/114585 PCT/US2009/036739 -15 equivalents, as described above. The DNA deletions and recombinations described in this section may be carried out by well-known methods. Another source of DNA is a phage display library of antibodies, as is known in the art. The exemplified antibodies of the current invention were made via hybridoma technology from immunized mice. 5 Additionally, the present invention provides expression vectors containing the polynucleotide sequences previously described operably linked to an expression sequence, a promoter and an enhancer sequence. A variety of expression vectors for the efficient synthesis of antibody polypeptide in prokaryotic systems, such as bacteria, and eukaryotic systems, including but not limited to yeast and mammalian cell culture 10 systems, have been developed. The vectors of the present invention can comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences. Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids from . coli, such as colE1, pCR1, pBR 322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and 15 other filamentous single-stranded DNA phages. An example of a vector useful in yeast is the 2pt plasmid. Suitable vectors for expression in mammalian cells include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and shuttle vectors derived from combination of functional mammalian vectors, such as those described above, and functional plasmids and phage DNA. 20 Additional eukaryotic expression vectors are known in the art (e.g., P.J. Southern and P. Berg, J. Mol. Apple. Genet. 1: 327-341 (1982); Subramani et al., Mol. Cell. Biol. 1: 854-864 (1981); Kaufmann and Sharp, J. Mol. Biol. 159: 601-664 (1982); Scahill et al., Proc. Nati. Acad. Sci. USA 80: 4654-4659 (1983); and Urlaub and Chasin, Proc. NatL. Acad. Sci. USA 77: 4216-4220 (1980). 25 The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major 30 operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the WO 2009/114585 PCT/US2009/036739 -16 promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations 5 thereof. The present invention also provides recombinant host cells containing the expression vectors previously described. Antibodies of the present invention can be expressed in cell lines other than in hybridomas. Nucleic acids, which comprise a sequence encoding a polypeptide according to the invention, can be used for 10 transformation of a suitable mammalian host cell. Cell lines of particular preference are selected based on high level of expression, constitutive expression of protein of interest and minimal contamination from host proteins. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines, such as but not limited to, NSO cells 15 (non-secreting (0) mouse myeloma cells), mouse myeloma cells, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells and many others. Suitable additional eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRCl, Pseudomonas, Bacillus, such as Bacillus 20 subtilis, and Streptomyces. These present recombinant host cells can be used to produce an antibody, or fragment thereof, by culturing the cells under conditions permitting expression of the antibody or fragment thereof and purifying the antibody or fragment thereof from the host cell or medium surrounding the host cell. Targeting of the expressed antibody or 25 fragment for secretion in the recombinant host cells can be facilitated by inserting a signal or secretory leader peptide-encoding sequence (see, Shokri et al., (2003) Appl Microbiol Biotechnol. 60(6):654-64, Nielsen et al., Prot. Eng. (1997) 10:1-6 and von Heinje et al., (1986) Nucl. Acids Res. 14:4683-4690) at the 5' end of the antibody-encoding gene of interest. These secretory leader peptide elements can be derived from either prokaryotic 30 or eukaryotic sequences. Accordingly suitably, secretory leader peptides are used, being WO 2009/114585 PCT/US2009/036739 -17 amino acids joined to the N-terminal end of a polypeptide to direct movement of the polypeptide out of the host cell cytosol and secretion into the medium. The antibodies of this invention can be fused to additional amino acid residues. Such amino acid residues can be a peptide tag, perhaps to facilitate isolation. Other 5 amino acid residues for homing of the antibodies to specific organs or tissues are also contemplated. Another embodiment for the preparation of antibodies in the present invention is the expression of the nucleic acid encoding the antibody according to the invention in a transgenic animal that has a substantial portion of the human antibody producing genome 10 inserted and is rendered deficient in the production of endogenous antibodies. Transgenic animals include, but are not limited to mice, goat, and rabbit. One further embodiment of the invention includes expression of the antibody-coding gene in, for example, the mammary gland of the animal for secretion of the polypeptide during lactation. A method of treating tumor growth in a mammal by administering to the mammal 15 an effective amount of an antibody is also provided by the present invention. Suitable tumors to be treated according to the present invention preferably express TYRP 1. While not intended to be bound to any particular mechanism, the present methods provide for treatment of the growth of cancer cells including malignant melanoma. "Treatment" or "treat", in the context of the present invention refers to therapeutic treatment including 20 inhibiting, slowing, lessening or reversing the progress of the underlying condition or undesired physiological change associated with a disease or disorder, ameliorating clinical symptoms of a condition or preventing the appearance of clinical symptoms of the condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or disorder, stabilization 25 of a disease or disorder (i.e., where the disease or disorder does not worsen), delay or slowing of the progression of a disease or disorder, amelioration or palliation of the disease or disorder, and remission (whether partial or total) of the disease or disorder, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment 30 include those already with the disease. In one embodiment, the present invention can be used as a medicament.

WO 2009/114585 PCT/US2009/036739 -18 The term "melanoma" includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, modular melanoma, lentigo malignant melanoma, acral 5 lentiginous melanoma, invasive melanoma and familial atypical mole and melanoma (FAM-M) syndrome. In one embodiment of the invention, melanoma is a specific form of cancer. In another embodiment the melanoma can be malignant. One embodiment of the present invention would be to utilize, as described below, the presently described anti TYRP1 antibodies to treat first line melanoma. In another embodiment, the presently 10 described anti-TYRPI antibodies would be first line treatment for metastatic melanoma, that is, they would be used in a first round of treatment of newly diagnosed metastatic melanoma. In the methods of the present invention, a therapeutically effective amount of an antibody of the invention is administered to a mammal in need thereof Effective doses of 15 the compositions of the present invention, for treatment of disorders as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. The term administering as used herein means delivering the antibodies of the present invention to a 20 mammal by any method that can achieve the result sought. They can be administered, for example, intravenously or intramuscularly. Although human antibodies of the invention are particularly useful for administration to humans, they can be administered to other mammals as well. The term mammal as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets and farm animals. Therapeutically 25 effective amount means an amount of antibody of the present invention that, when administered to a mammal, is effective in producing the desired therapeutic effect, such as inhibiting tumor growth. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy. The pharmaceutical compositions of the invention may include a "therapeutically 30 effective amount" of an anti-TYRP1 antibody of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time WO 2009/114585 PCT/US2009/036739 -19 necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any 5 toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). The identification of such disease is well within the ability and knowledge of one skilled in the art. For example, human individuals who are suffering from malignant 10 melanoma or who are at risk of developing clinically significant symptoms are suitable for administration of the present anti-TYRP 1 antibodies. The present anti-TYRP1 antibodies are administered for therapeutic treatments to a patient suffering from malignant melanoma in an amount sufficient to inhibit, or reduce the progression of the tumor or pathologic condition. Progression includes, e.g., the 15 growth, invasiveness, metastases and/or recurrence of the tumor or pathologic condition. An amount adequate to accomplish this is defined as a therapeutically effective dose. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system. Dosing schedules will also vary with the disease state and status of the patient, and will typically range from a single bolus 20 dosage or continuous infusion to multiple administrations per day (e.g., every 4-6 hours), or as indicated by the treating physician and the patient's condition. An exemplary, non limiting range for a therapeutically effective amount of an antibody of the invention is 0.1-50 mg/kg, more preferably 3-35 mg/kg, and more preferably 5-20 mg/kg. Dosing amounts and frequencies will be determined by the physicians treating the patient and 25 may include doses from less than 1 mg/kg to over 100 mg/kg given daily, three times per week, weekly, once every two weeks, or less often. Dose per administration may be in the range of 1-100, 2-75, or 5-60 mg/kg. It should be noted, however, that the present invention is not limited to any particular dose. In an embodiment of the invention, anti-TYRP 1 antibodies can be administered in 30 combination with one or more other anti-neoplastic agents. Any suitable anti-neoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof WO 2009/114585 PCT/US2009/036739 -20 The anti-neoplastic agent can be an alkylating agent or an anti-metabolite. Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine (DTIC). In vivo studies indicate that administering 20D7 in combination with dacarbazine (DTIC) demonstrate stronger anti-tumor activity compared to mono 5 therapy on a human 624mel xenograft. In one embodiment of the present invention, the presently described anti-TYRP1 antibodies are given in combination with dacarbazine. Examples of anti-metabolites include, but are not limited to, doxorubicin, daunorubicin, paclitaxel, irinotecan (CPT- 1l), and topotecan. When the anti-neoplastic agent is radiation, the source of the radiation can be either external (external beam radiation 10 therapy - EBRT) or internal (brachytherapy - BT) to the patient being treated. The dose of anti-neoplastic agent administered depends on numerous factors, including, for example, the type of agent, the type and severity of the tumor being treated and the route of administration of the agent. It should be emphasized, however, that the present invention is not limited to any particular dose. 15 In the present invention, any suitable method or route can be used to administer anti- TYRP1 antibodies of the invention, and optionally, to co-administer anti-neoplastic agents and/or antagonists of other receptors. The anti-neoplastic agent regimens utilized according to the invention, include any regimen believed to be optimally suitable for the treatment of the patient's neoplastic condition. Different malignancies can require use of 20 specific anti-tumor antibodies and specific anti-neoplastic agents, which will be determined on a patient to patient basis. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Parenteral routes are preferred. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration. 25 In another aspect of the invention, an anti-TYRP1 antibody of the invention can be chemically or biosynthetically linked to one or more anti-neoplastic or anti-angiogenic agents. The invention further contemplates anti-TYRP1 antibodies linked to target or reporter moieties, including by way of example only anti-neoplastic agents, other 30 antibodies or reporters, such as radiolabled isotopes, in a diagnostic system where a detectable signal-producing agent is conjugated to the antibody.

WO 2009/114585 PCT/US2009/036739 -21 It is understood that the anti-TYRPI antibodies of the invention, where used in a mammal for the purpose of prophylaxis or treatment, will be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, 5 phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the injection can, as is well known in the art, be formulated so as to 10 provide quick, sustained or delayed release of the active ingredient after administration to the mammal. Moreover, included within the scope of the present invention is use of the present antibodies in vivo and in vitro for investigative or diagnostic methods, which are well known in the art. The diagnostic methods include kits, which contain antibodies of the 15 present invention. In one embodiment, the invention is a human monoclonal antibody, or fragment thereof, specific for TYRPl. In another embodiment, the invention is a chimeric monoclonal antibody, or fragment thereof, specific for TYRPl. In another embodiment the CDR regions of the antibody are identical to the CDR regions of CTA99. In a 20 different embodiment the CDR regions of the antibody are identical to the CDR regions of 20D7 or 20D7S. In one embodiment, the antibody binds to TYRP 1 with a dissociation rate constant (Kd or kof between 1.7 x 10- 4 1/s (sec- 1 , 1/seconds) and 5 x 10- 4 1/s, as measured by surface plasmon resonance, described herein, at ambient laboratory temperature (20 0 C 25 25 0 C). In another embodiment, the antibody binds to TYRPI with a Kd or koff between 1.7 x 10- 4 1/s and 3.5 x 10- 4 1/s. In a further embodiment, the antibody binds to TYRPI with a dissociation rate constant, as measured by surface plasmon resonance, that is within 10% of the dissociation rate constant determined for 20D7, 20D7S, or CTA99 under the same conditions.

WO 2009/114585 PCT/US2009/036739 -22 One embodiment of the present invention comprises a monoclonal antibody, or fragment thereof, specific for TYRP1 comprising one or more complementarity determining regions (CDRs) selected from the group consisting of the CDRs in Tables 1 and 2. In another embodiment, the invention is a monoclonal antibody, or fragment 5 thereof, specific for TYRPI having a light chain CDR1 region with the sequence: RASQSVSSYLA (SEQ ID NO:4). In another embodiment, the invention is a monoclonal antibody, or fragment thereof, specific for TYRP1 having a heavy chain CDR3 with the sequence: RYSSSWYLDY (SEQ ID NO:3). In a different embodiment, the invention is a monoclonal antibody, or fragment thereof, comprising (i) a light chain variable region 10 selected from the group consisting of 20D7, 20D7S and CTA99 and (ii) a heavy chain variable region selected from the group consisting of 20D7, 20D7S and CTA99. In another embodiment, the invention is a monoclonal antibody, or fragment thereof, specific for TYRPI comprising (i) a light chain variable region of CTA99 (ii) a heavy chain variable region of CTA99, and (iii) human immunoglobulin GI (hIgG1) constant 15 regions. Another embodiment of this invention is a method of treating cancer in a mammal comprising administering to the mammal an effective amount of an antibody, or fragment thereof, of any of the embodiments already described. The invention also provides a method to treat a malignant melanoma. Another treatment method provided by this 20 invention combines using the antibodies or fragments thereof of this invention along with administering an additional anti-cancer agent or treatment. In one treatment method, the anti-cancer agent is dacarbazine (DTIC). It is to be understood and expected that variations in the principles of invention herein disclosed can be made by one skilled in the art and it is intended that such 25 modifications are to be included within the scope of the present invention. All references mentioned herein are incorporated in their entirety. EXAMPLES The examples that follow further illustrate the invention, but should not be 30 construed to limit the scope in any way. They should in no way be construed, however, as limiting the broad scope of the invention. Detailed description of conventional WO 2009/114585 PCT/US2009/036739 -23 methods, such as those employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, the introduction of plasmids into host cells, and the expression and determination thereof of genes and gene products can be obtained from numerous publications, including Sambrook, J. et al., 5 (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press. Animals and Cell Lines Maintain SKmel28, SKmel23, 624mel, 1102mel and A375 in RPMI 1640 10 (Invitrogen Life Technologies) with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT) and routinely test for Mycoplasma contamination. SKmel23 and SKmel28 were provided by Dr. Alan Houghton (Memorial Sloan-Kettering Cancer Center, New York, NY). 624mel and 1102mel were obtained from Dr. Steve Rosenberg (National Cancer Institute, Bethesda, MD). A375 was purchased from American Type 15 Culture Collection (Manassas, VA). Six to eight week old female Nu/Nu mice were purchased from Taconic Farms (Germantown, NY). Expression and Purification of Human and Chimeric Anti-TYRP 1 Antibodies For each antibody, engineer a suitable heavy chain nucleotide sequence, for 20 example SEQ ID NOs 21, 22, or 23 (for 20D7, 20D7S and CTA99 respectively) into a suitable expression plasmid, for example pGSHC, and engineer a suitable light chain nucleotide sequence, for example SEQ ID No. 26 or 27 (for 20D7/20D7S and CTA99 respectively) into a suitable expression plasmid, such as pGSLC, by a suitable method such as PCR cloning. To establish a stable cell line, co-transfect in a suitable host cell 25 line, such as NSO cells, with linearized heavy and light chain plasmids by electroporation and culture in suitable media such as glutamine free Dulbecco's Modified Eagle Medium with dialyzed fetal calf serum and glutamine synthetase supplement. Screen clones for antibody expression by an enzyme-linked immunosorbent assay (ELISA) and select the highest producer for culture in spinner flasks. Purify antibodies by a suitable method such 30 as protein-A affinity chromatography.

WO 2009/114585 PCT/US2009/036739 -24 One embodiment of the present invention is the recombinant human monoclonal antibody 20D7, a full length IgGiK targeting cell surface expressed tyrosinase-related protein-1 (TYRPi or TRPi). The antibody is comprised of a human gamma-1 heavy chain (HC) (subgroup I) and a human kappa light chain (subgroup III). 20D7 was shown 5 to selectively bind to human TYRPi with high affinity and mediated potent anti-tumor activity in xenograft models by a mechanism involving activation of immune effector function. One embodiment of the present invention is the recombinant human monoclonal antibody 20D7S, a full length IgGiK targeting cell surface expressed tyrosinase-related 10 protein-1 (TYRPi or TRPi). The antibody is comprised of a human gamma-1 heavy chain (HC) (subgroup I) and a human kappa light chain (subgroup III). 20D7S was created in an effort to generate an even more stable molecule; a free cysteine residue within the heavy chain variable region (C47) was converted to a serine residue by site directed mutagenesis. This unpaired or free cysteine has the potential to mis-pair with 15 other cysteines that participate in intra- and inter-chain disulfide bridging of heavy and light chain polypeptides. Mis-pairing could potentially result in improper folding and processing, increasing the heterogeneity of the product and potentially its stability. The SDS PAGE gel analysis described herein confirms the presence of free light chain and free heavy chain in the preparation of 20D7, but the presence of free light chains and free 20 heavy chains are reduced or eliminated in the preparation of 20D7S. Another embodiment of the present invention is CTA99, a chimeric antibody with a human constant region IgGi. Murine antibody TA99 (United States Patent 4,798,790) contains two light chains; one TA99 light chain is specific to TYRPi and the other is from parental mouse myeloma cells. There is decreased activity because the 25 contaminated light chain is unable to bind to TYRPl. CTA99 was constructed to remediate this flaw and thereby improve activity. In one embodiment of the present invention, CTA99 was designed with two identical heavy chains and two identical light chains. Studies described herein illustrate CTA99's increased activity and binding affinity, as well as effector functions more suitable for humans. 30 Tables I and 2 provide the amino acid sequences and SEQ ID NOs of the various CDRs of the present invention. Table 3 provides the SEQ ID NOs of the various WO 2009/114585 PCT/US2009/036739 -25 sequences related to the present invention. Polynucleic acid sequences that encode the amino acid sequences disclosed below are also included within the scope of the present invention. 5 Table 1: Amino Acid Sequence of 20D7 and 20D7S Antibody Heavy and Light Chain Variable Region CDRs. Heavy Chain SEQ ID Light Chain SEQ ID ___ __________ NO. _ ______ NO. CDR1 GYTFTSYAMN 1 RASQSVSSYLA 4 CDR2 WINTNTGNPTYAQGFTG 2 DASNRAT 5 CDR3 RYSSSWYLDY 3 QQRSNWLMYT 6 Table 2: Amino Acid Sequence of CTA99 Antibody Heavy and Light Chain Variable Region CDRs. Heavy Chain SEQ ID Light Chain SEQ ID _____NO. ___ ____ NO. CDR1 GFNIKDYFLH 7 RASGNIYNYLA 10 CDR2 WINPDNGNTVYDPKFQG 8 DAKTLAD 11 CDR3 DYTYEKAALDY 9 QHFWSLPFT 12 10 Table 3: Amino Acid Sequence SEQ. ID. NOs of 20D7, 20D7S, and CTA99 Antibodies Heavy Chain Light Chain Antibody With Variable Without With Variable Without signal region signal signal region signal 20D7 18 13 29 24 16 32 20D7S 19 14 30 24 16 32 CTA99 20 15 31 25 17 33 Antibodies used in experiments comprised full-length heavy and light chains without signals, as given in Table 3. 15 Table 4: Summary of Data from 20D7, 20D7S, and CTA99 Antibodies Competition ACS Affinity Human FcR Human Human Antibody ELISA (mel) KD (M) binding (M) ADCC CDC

(EC

50 ) (M) (() (%) TA99 4.0 x 10- 10 +++ 1.7 x 10- 9 ND ND ND WO 2009/114585 PCT/US2009/036739 -26 CTA99 1.1 x 10-10 +++ 1.1 x 10- 9 ND 48 94 20D7 1.1 x 10- 10 +++ 0.28 x 10- 9 1.1 x 10- 1 0 42 100 20D7S 0.98 x 10-10 + 0.28 x 10-9 0.97 x 10-10 56 ND ND = not determined. Competition Enzyme-Linked Immunosorbent Assay (ELISA) Coat falcon flexible 96-well flat-bottomed plates with recombinant human TYRP 5 (.5ug/mL x 50 ptL) at 4' Celsius overnight. The next day, block the plate with 5% FBS in PBS containing 0.1% Tween-20 for 2 h at room temperature. Add various amounts of antibodies in 100 ptL samples. Wash the plate 3x with PBS/Tween and add 100 ptL of horseradish peroxidase-conjugated goat anti-human antibody (Biosource, Camarillo, CA) diluted at 1:5000 in 100 ptL to the plate and incubate for one hour at room temperature (20 10 - 25 C). Wash the plate 3x and add 50 ptL /well of 3, 3', 5, 5'-tetramethylbenzidine (TMB; KPL, Gaithersburg, MD) substrate to the plate. Read the plates at 450 nm using a microplate reader (such as Molecular Devices). Table 5: Enzyme-Linked Immunosorbent Assay (ELISA) Binding to TYRP 1 Antibody

(EC

5 0 ) (M) TA99 4.0 x 10-10 CTA99 1.1 x 10-10 20D7 1.1 x 10-10 20D7S 0.98 x 10-10 15 The half minimal effect concentration (EC 5 0) is measured in Molar (M). Antibodies, including human 20D7, human 20D7S and CTA99, exhibit specific binding to human TYRPI in an ELISA assay. Flow Cytometry 20 Treat 624mel cells for 1 hour on ice with either 5 ptg/mL human IgG, or anti TYRPI MAbs in 1%BSA/PBS. Wash cells 3x in 1% BSA/PBS and incubate for 1 hour with fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG. Wash cells and WO 2009/114585 PCT/US2009/036739 -27 analyze by a suitable flow cytometer, such as Epics XL Flow Cytometer (Coulter). Flow cytometry analysis shows that 20D7S antibodies as exemplified herein exhibit binding to native TYRPI expressing in human cell lines SKmel28 and SKmel23 as compared to control human IgG 1 . Similarly, flow cytometry analysis shows that for CTA99 and 20D7 5 antibodies exemplified herein exhibit binding to native TYRP1 expressing human cell line 624mel as compared to control human IgG 1 . Surface Plasmon Resonance / Biacore Analysis Measure the binding kinetics of the antibodies to recombinant human TYRP1 at 10 ambient laboratory temperature (20 'C - 25 'C) using the surface plasmon resonance, for example a Biacore biosensor (Pharmacia). Immobilize TYRPI protein onto a CM5 research grade sensor chip and inject the antibodies at concentrations ranging from 0.5 nM to 100 nM. Acquire sensorgrams for each concentration and evaluate using the BIA Evaluations 3.2 program to determine the rate constants kon and koff Kd, also referred to 15 as kog is the rate constant of the dissociation reaction. Ka, also referred to as kon, is the rate constant of the association reaction. KD is a measure of binding affinity; calculate KD from the ratio of the rate constants koff/ kon measured in Molar (M). Ka, the Kd, and KD for the antibodies exemplified herein, TA99, CTA99, 20D7, and 20D7S, are summarized below in Table 6. 20 Table 6: Binding Kinetics of Antibodies to Recombinant Human TYRPI Antibody Ka (1/Ms) Kd (1/s) KD(M) kon koff TA99 9.5 x 10 4 1.6 x 10- 4 1.7 x 10-9 CTA99 2.8 x 10 4 3.0 x 10- 4 1.1 x 10-9 20D7 6.8 x 10 5 1.9 x 10- 4 0.28 x 10-9 20D7S 6.4 x 10 5 1.8 x 10- 4 0.28 x 10-9 Biacore analysis of the binding of 20D7 and 20D7S to human TYRPI demonstrate substantial specific binding affinities; accordingly 20D7 and 20D7S are valid 25 candidates for therapeutic antibodies.

WO 2009/114585 PCT/US2009/036739 -28 Complement Dependent Cytotoxicity (CDC) Assay Wash human melanoma cell line 624mel 3 times and bring to a concentration of 106 viable cells/mL in a suitable media, such as AIM V Media (Invitrogen Life 5 Technologies). Plate one hundred microliters of cells in 96-well, round-bottom Falcon plates and incubate with MAb 20D7 or hIgG (Jackson Immunoresearch, West Grove, PA) starting at 3.7 Vtg/mL diluted down 1:3, for 1 hr at 37 'C. Add low-Tox-M Rabbit complement (Cedarlane Labs, Westbury, NY) diluted 1:5 in AIM V at 50 1d/well and incubate for 1hr at 37'C. Count cells for viability with Trypan Blue (Invitrogen Life 10 Technologies). Table 7 shows the percentage of cytotoxicityat various antibody concentrations in the CDC assay. The data demonstrate that 20D7 and CTA99 antibodies induce CDC against TYRPI (+) human 624mel cells in vitro. 20D7 triggered dose-dependent complement-mediated cell lysis of 624mel cells, reaching complete cell lysis in this assay 15 with an antibody concentration of 3.7 Vtg/mL. Accordingly, there is a strong immune effector response for CDC in 20D7 and CTA99. % Specific Lysis = Test % Cytotoxicity - Negative Control % Cytotoxicity. Table 7: CDC Data Demonstrating Complement Activation by 20D7 and CTA99 Antibody Concentration CTA99 20D7 (Vtg/mL) 3.7 95% 100% 1.23 54% 84% 0.41 53% 38% 0.14 17% 35% 0.05 9.5% 24% 20 Antibody Dependent Cellular Cytotoxicity (ADCC) against Human Melanoma In Vitro Collect and wash 624mel cells with a suitable media, such as AIM V media (Invitrogen Life Technologies) and plate the cells at a density of 10,000 cells /well in a 100 VtL volume, in a 96-well Falcon U bottom plate. Add antibodies at 5 ptg/mL in a 50 25 ptL volume and incubate at 37 'C for 0.5 hr with target cells. Effector cells were added in WO 2009/114585 PCT/US2009/036739 -29 a volume of 50 jiL at various E:T (effector:target) ratios. Plates were further incubated for 4 hrs at 37'C. After incubation, plates were spun down at 800 g, and 100 XL of supernatants were gently transferred to 96 well flat bottom plates. Lactate dehydrogenase assay reagent was added as specified by the manufacturer (Roche) and plates were read at 5 490 nm. Controls in assay: Target Spontaneous and Target Maximum (by adding 50 tL of 4% triton). Lysis is dependent on the effector to target concentration with lysis of 50% of target cells occurring at an E:T ratio of 100:1. At a fixed concentration (5 ptg/mL) 20D7 and CTA99 activate the lysing of 624mel cells. Table 8 shows the percentage of cell 10 cytotoxicity in the presence of various effector cell to tumor cell ratios (E:T Ratio) in the ADCC assay. 20D7S, 20D7 and CTA99 antibodies induce ADCC against TYRPI (+) human 624mel cells in vitro. There is a strong immune effector response for ADCC in 20D7S, 20D7 and CTA99. % Cytotoxicity = (Experimental - target spontaneous) / 15 (Target Maximum - target spontaneous) X 100 Table 8: ADCC against Human Melanoma In Vitro % Cytotoxicity E:T Ratio 20D7 20D7S CTA99 100:1 43% 56% 49% 50:1 33% 47% 39% 25:1 31% 43% 17% 12.5:1 17% 23% 12% 20D7S and 20D7 Stability Assays 20 Load 20D7 and 20D7S into SDS-PAGE gel (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Run gels using BioRad apparatus until the bromophenol blue dye is just off. Visualize the separated proteins using Coomassie Dye. In the SDS PAGE analysis, more stable molecules are seen as a single band with few if any free light and or heavy chains; the presence of free light chains and free heavy 25 chains are evidence of less stable molecules. The SDS PAGE gel of 20D7 showed the presence of obvious free light and heavy chains. The SDS PAGE gel of 20D7S showed WO 2009/114585 PCT/US2009/036739 -30 very few free light and heavy chains. Accordingly, 20D7S is a more stable IgG1 molecule than 20D7. CTA99 and 20D7 Effectively Treat Human Melanoma Xenografts. 5 For the following subcutaneous studies, tumor volumes are calculated by the formula [xT/6(W1xW2xW2)], where W1 represents the largest tumor diameter and W2 represents the smallest tumor diameter. %T/C = 100 x (Treatment Volume/Initial Volume) / (Control Volume/Initial Volume). Statistical analysis is done using traditional p-value techniques. For the subcutaneous studies the p value is calculated based on the 10 tumor volume of animals receiving the anti-TYPR antibody verses the tumor volume of the control animals. For the following metastatic studies, tumor inhibition is measured by counting lung surface nodules. % inhibition = 100 x (control nodules# - treatment nodules#) / (control nodules#). Statistical analysis is done using traditional p-value techniques. For the metastatic studies the p-value is calculated based on the nodules 15 observed in the animals receiving the anti-TYPR antibody verses the nodules observed in the control animals. In vivo single agent activity of anti-TYRPI antibody on xenograft models of human melanoma 20 Harvest, wash and resuspend 624mel cultured cells in a 50/50 solution of Matrigel and RPMI 1640 media (10% FBS heat inactivated). For the subcutaneous tumor model, inject 2 x 10 6 cells subcutaneously into the left flank of nude mice. When tumors reach 200 mm 3 , treat mice with anti-TYRPI antibodies or control human IgG; administer antibodies 1 mg/mouse three times per week. Measure tumors with calipers twice a week 25 and calculate % T/C. For the in vivo single agent activity of anti-TYRP1 antibody on xenograft models of human melanoma, growth of SKmel28 xenografts was inhibited by 20D7 treatment compared to human IgG control (T/C = 51%; P = 0.01 at day 43). Established 624mel tumors were also shown to be inhibited significantly by 20D7 treatment. Tumor growth was inhibited and reached statistical significance at day 16 post 30 initiation of antibody treatment (T/C = 44%; P = 0.01). Additional human melanoma xenografts were evaluated for anti-tumor activity of 20D7. Cell lines A375 and 1102mel WO 2009/114585 PCT/US2009/036739 -31 were shown to be significantly inhibited by single agent 20D7 11 days post antibody treatment (T/C = 42%; P = 0.01 and T/C = 43%; P = 0.004 for A375 and 1102mel respectively). Lysates of skins from different species are incubated with TA99 (5 pXg/ml) for 2 hours at room temperature. The lysates are then precipitated with protein A and 5 subjected to SDS-PAGE under reducing and non-reducing conditions using four 12% gradient gels. After electrophoresis, the gels are transferred to PVDF membrane (Invitrogen Life Technologies). The membrane is probed with 20D7S (5 pXg/ml) followed by an HRP labeled anti-human IgG (Zymed, South San Francisco, CA). The blot is developed using a chemiluminescent substrate (KPL, Gaithersburg, MD). The data show 10 that 20D7 readily cross-reacts with mouse TYRPl. However, no overt toxicity was apparent in any animals treated with 20D7. Body weight and overall appearance were not significantly different in 20D7 treated animals relative to human IgG control treated mice. In vivo single agent dose response activity of anti-TYRPI antibody on xenograft models 15 of human melanoma Mix Skmel28 cells in a 50/50 solution of Matrigel and RPMI 1640 media (10% FBS heat inactivated). Inject 2 x 10 6 cells subcutaneously into the left flank of nude mice. When tumors reach 200 mm 3 , treat mice with anti-TYRPI antibodies (6 mg/kg, 20 mg/kg, or 60 mg/kg) or control human IgG three times per week. Measure tumors with 20 calipers twice a week and calculate % T/C. The dose-response study of 20D7 on SKmel28 xenografts indicated a dose-dependent anti-tumor response. Even at 6 mg/kg dose, tumors were significantly inhibited by 20D7 (T/C = 69%; P < 0.0001). The anti tumor effects at each dose were statistically significant: 6 mg/kg and 20 mg/kg (T/C = 50%; P = 0.04), 6 mg/kg and 60 mg/kg (T/C = 19%; P < 0.003). 25 In vivo single agent activity of anti-TYRP 1 antibody in two metastatic melanoma models B16BL6 is an aggressive and spontaneously arising murine melanoma. It forms lung metastases in nude mice after intravenous administration. Harvest, wash and resuspend cultured SKmel23 and B16BL6 cells melanoma cells in RPMI 1640 media 30 (10% FBS heat inactivated).

WO 2009/114585 PCT/US2009/036739 -32 Model 1: Inject 1 x 10 5 B16BL6 cells intravenously. On the second day after the tumor injection, administer to mice anti-TYRP1 antibodies or control human IgG, according to three different dose concentrations (200 jig/mouse, 500 jig/mouse, and 1 mg/mouse). Sacrifice mice on day 20, remove lungs, count lung surface nodules and 5 calculate percent inhibition. Heavy metastases are detected over the surface of the lungs in human IgG treated mice; significantly fewer metastases are noted in 20D7 treated animals. All three concentrations of 20D7 reduce the level of lung metastases (Inhibition - 65%, 74%, and 95%, respectively). Model 2: Inject 1 x 10 5 human SKmel23 cells intravenously. On the second day 10 after the tumor injection, administer to mice anti-TYRP1 antibodies or control human IgG, according to two different dose concentrations (200 jig/mouse and 500 jig/mouse). Sacrifice mice on day 20, remove lungs, count lung surface nodules and calculate percent inhibition. Metastatic nodules are significantly reduced by treatment with 20D7 or CTA99 at 200 jig/dose and at 500 jig/dose. 20D7 reduced metastasis by 58% and 73% 15 respectively. CTA99 reduced metastasis by 63% and 75% respectively. These results demonstrate that in two separate models, melanoma metastasis is inhibited by 20D7 and CTA99. In vivo comparative studies of 20D7 and 20D7S inhibition of tumor growth on 20 subcutaneous xenograft and metastatic models of human melanoma For the subcutaneous tumor model, harvest, wash and resuspend 624mel cultured cells in a 50/50 solution of Matrigel and RPMI 1640 media (10% FBS heat inactivated), then inject 2 x 10 6 624mel cells subcutaneously into the left flank of nude mice. When tumors reach 200 mm 3 , treat mice with 20D7 or 20D7S, 40 mg/kg twice per week. 25 Measure tumors with calipers twice a week and calculate % T/C. In the 624mel subcutaneous xenograft model, 20D7 inhibited tumor growth T/C =21% and for 20D7S T/C =25%. Both 20D7 and 20D7S inhibited tumor growth in the xenograft model. For the metastatic model, harvest, wash and resuspend 888mel cultured cells in RPMI 1640 media (10% FBS heat inactivated), inject 888mel cells intravenously. On the 30 second day after the tumor injection, administer to mice anti-TYRPI antibodies or control WO 2009/114585 PCT/US2009/036739 -33 human IgG. Sacrifice mice on day 20, remove lungs, count lung surface nodules and calculate percent inhibition. In the metastatic model of 888mel in nude mice, both 20D7 and 20D7S significantly inhibited lung surface metastasis: 20D7 inhibition = 77%, p=0.0005; 20D7S inhibition = 80%, p=0.0005. Both 20D7 and 20D7S reduced 5 metastasis of melanoma in the metastatic model. 20D7 and Dacarbazine (DTIC) combination treatment demonstrated stronger anti-tumor activity compared to mono-therapy on human xenograft For the subcutaneous model, harvest, wash and resuspend cultured 624mel cells, 10 in a 50/50 solution of Matrigel and RPMI 1640 media (10% FBS heat inactivated). Inject 2 x 10 6 624mel cells into the left flank of nude mice. When tumors reach 200 mm 3 , treat mice with anti-TYRP 1 antibodies, DTIC, or a combination of anti-TYRP 1 antibodies and DTIC. Administer 40 mg/kg antibodies once per week. Administer DTIC 5 mg/kg once per week. Measure tumors with calipers twice a week and calculate % T/C. For 15 metastatic model, harvest, wash and resuspend cultured SKmel23, 888mel and B16 melanoma cells, in RPMI 1640 media (10% FBS heat inactivated). Inject SKmel23, 888mel and B16 melanoma cells intravenously. On the second day after the tumor injection, administer to mice anti-TYRPI antibodies or control human IgG. Sacrifice mice on day 20, remove lungs, count lung surface nodules and calculate percent 20 inhibition. As demonstrated in the subcutaneous model, 20D7 and DTIC combination treatment inhibited tumor growth significantly better than 20D7 (p<0.001) or DTIC (p<0.001) alone. 25 Table 9: Summary of anti-tumor Activities of 20D7 In Vivo Model Tumor Measurement 20D7 20D7+DTIC Metastatic SKmel23 % inhibition 80% 99%* Metastatic 888 mel % inhibition 77% 96%* Metastatic B16 % inhibition 95% ND Subcutaneous 624mel % T/C 500 190 ___________ __________ ___________ P-0.004 P<0.0001 WO 2009/114585 PCT/US2009/036739 -34 Table 9 summarizes in vivo anti-tumor activities of 20D7S compared to monotherapy 20D7 or DTIC in 4 models. The anti-tumor activities of 20D7 in vivo are shown by percent inhibition in metastatic models (the * denotes statistical significance; ND denotes not determined. The anti-tumor activities of 20D7 in vivo are shown by 5 percent T/C in subcutaneous models. Treatment with 20D7 and Dacarbazine (DTIC) in combination demonstrated stronger anti-tumor activity as compared to mono-therapy.

Claims

2. The antibody or fragment thereof of claim 1, comprising a VL comprising to the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWLMYT FGQGTKLEIK (SEQ ID NO: 16); and a VH comprising the amino acid sequence: is QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLESMGWINT NTGNPTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRYSSSWYLDY WGQGTLVTVSS (SEQ ID NO: 14).
3. The antibody or fragment thereof of claim 1 or claim 2 comprising a heavy chain of SEQ ID NO: 30 and a light chain of SEQ ID NO: 32. 20 4. An antibody or fragment thereof as claimed in any one of claims 1-3 comprising two heavy chains of SEQ ID NO: 30 and two light chains of SEQ ID NO: 32.
5. An isolated polynucleic acid comprising a nucleotide sequence encoding the antibody or fragment according to any one of claims 1 to 4.
6. An expression vector comprising the polynucleic acid of claim 5 operably 25 linked to expression control elements such that the encoded antibody or fragment may be expressed.
7. A recombinant cell comprising the expression vector of claim 6, which recombinant cell is capable of producing an antibody or fragment according to any one of claims 1-4. 30 8. An antibody or fragment produced by culturing the recombinant cell according to claim 7 so that the antibody or fragment is produced and recovering the antibody or fragment from the culture.
9. A pharmaceutical composition comprising an antibody or fragment as claimed in any one of claims 1 to 4 or 8 together with a pharmaceutically acceptable 35 carrier, diluent or excipient.
10. An antibody or fragment as claimed in any one of claims 1 to 4 or 8 for use as a medicament.
11. An antibody or fragment as claimed in any one of claims I to 4 or 8 for use in the treatment of cancer. 36
12. An antibody or fragment as claimed in claim 11, wherein the cancer is malignant melanoma.
13. The monoclonal antibody or fragment thereof of claim 1, substantially as hereinbefore described with reference to any one of the examples. 5 14. Use of an antibody or fragment thereof as claimed in any one of claims I to 4, 8 or 13 for the manufacture of a medicament for treating or preventing a condition in a subject indicating administration of said antibody or fragment thereof.
15. Use of an antibody or fragment thereof as claimed in any one of claims I to 4, 8 or 13 for the manufacture of a medicament for treating cancer in a subject. 1o 16. The use as claimed in claim 15, wherein the cancer is malignant melanoma.
17. A method for treating or preventing a condition in a subject indicating administration of a TYRP I-binding antibody or fragment thereof, said method comprising administering to said subject an antibody or fragment thereof as claimed in any one of claims I to 4, 8 or 13.
18. A method for treating a cancer in a subject, said method comprising administering to said is subject an antibody or fragment thereof as claimed in any one of claims I to 4, 8 or 13.
19. The method as claimed in claim 18, wherein the cancer is malignant melanoma. ImClone LLC Patent Attorneys for the Applicant/Nominated Person 20 SPRUSON & FERGUSON 961908Seq.TXT SEQUENCE LISTING <110> ImClone LLC <120> ANTI-TYRP1 ANTIBODIES <130> X18551 <150> 61/069199 <151> 2008-03-12 <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Ala Met Asn 1 5 10 <210> 2 <211> 17 <212> PRT <213> Homo sapiens <400> 2 Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe Thr 1 5 10 15 Gly <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Homo sapiens <400> 4 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Homo sapiens Page 1 961908Seq.TXT <400> 5 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <400> 6 Gln Gln Arg Ser Asn Trp Leu Met Tyr Thr 1 5 10 <210> 7 <211> 10 <212> PRT <213> Mus musculus <400> 7 Gly Phe Asn Ile Lys Asp Tyr Phe Leu His 1 5 10 <210> 8 <211> 17 <212> PRT <213> Mus musculus <400> 8 Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Asp Pro Lys Phe Gln 1 5 10 15 Gly <210> 9 <211> 11 <212> PRT <213> Mus musculus <400> 9 Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> Mus musculus <400> 10 Arg Ala Ser Gly Asn Ile Tyr Asn Tyr Leu Ala 1 5 10 <210> 11 <211> 7 <212> PRT Page 2 961908Seq.TXT <213> Mus musculus <400> 11 Asp Ala Lys Thr Leu Ala Asp 1 5 <210> 12 <211> 9 <212> PRT <213> Mus musculus <400> 12 Gln His Phe Trp Ser Leu Pro Phe Thr 1 5 <210> 13 <211> 119 <212> PRT <213> Homo sapiens <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Cys Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 14 <211> 119 <212> PRT <213> Homo sapiens <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Page 3 961908Seq.TXT Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Ser Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 15 <211> 122 <212> PRT <213> Mus musculus <400> 15 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Leu Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Phe Leu His Trp Val Arg Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Thr Ala Ser Leu Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Ser Gly Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Ala Ser Val Ile Val Phe Ser Ala 115 120 Page 4 961908Seq.TXT <210> 16 <211> 108 <212> PRT <213> Homo sapiens <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Leu Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> Mus musculus <400> 17 Ala Ile Gln Met Ser Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro His Leu Leu Val 35 40 45 Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Ser Leu Gln Thr 65 70 75 80 Glu Asp Ser Gly Asn Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 Page 5 961908Seq.TXT <210> 18 <211> 468 <212> PRT <213> Homo sapiens <400> 18 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Cys Met Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala 65 70 75 80 Gln Gly Phe Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Page 6 961908Seq.TXT Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 <210> 19 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct Page 7 961908Seq.TXT <400> 19 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Ser Met Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala 65 70 75 80 Gln Gly Phe Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Page 8 961908Seq.TXT 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 <210> 20 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Page 9 961908Seq.TXT Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30 Pro Gly Ala Leu Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Asn Ile 35 40 45 Lys Asp Tyr Phe Leu His Trp Val Arg Gln Arg Pro Asp Gln Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Asp 65 70 75 80 Pro Lys Phe Gln Gly Thr Ala Ser Leu Thr Ala Asp Thr Ser Ser Asn 85 90 95 Thr Val Tyr Leu Gln Leu Ser Gly Leu Thr Ser Glu Asp Thr Ala Val 100 105 110 Tyr Phe Cys Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp 115 120 125 Tyr Trp Gly Gln Gly Ala Ser Val Ile Val Phe Ser Ala Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Page 10 961908Seq.TXT Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 21 <211> 1407 <212> DNA <213> Homo sapiens <400> 21 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattcacag 60 gtgcagctgg tccaatctgg gtctgagttg aagaagcctg gggcctcagt gaagatttcc 120 tgcaaggctt ctggatacac cttcactagc tatgctatga attgggtgcg acaggcccct 180 ggacaagggc ttgagtgtat gggatggatc aacaccaaca ctgggaaccc aacgtatgcc 240 cagggcttca caggacggtt tgtcttctcc atggacacct ctgtcagcac ggcatatctg 300 cagatcagca gcctaaaggc tgaggacact gccatatatt actgtgcgcc ccgatatagc 360 agcagctggt accttgatta ctggggccag ggaaccctgg tcaccgtgtc ctcagctagc 420 Page 11 961908Seq.TXT accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 480 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 540 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 660 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga gcccaaatct 720 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtatgtg 900 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 960 taccgtgtgg tcagcgtcct caccgtcctg caccaagact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1140 aagaaccaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 tccgacggct ccttcttcct ctattccaag ctcaccgtgg acaagagcag gtggcagcag 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1380 agcctctccc tgtctccggg caaatga 1407 <210> 22 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 22 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattcacag 60 gtgcagctgg tccaatctgg gtctgagttg aagaagcctg gggcctcagt gaagatttcc 120 tgcaaggctt ctggatacac cttcactagc tatgctatga attgggtgcg acaggcccct 180 ggacaagggc ttgagtctat gggatggatc aacaccaaca ctgggaaccc aacgtatgcc 240 cagggcttca caggacggtt tgtcttctcc atggacacct ctgtcagcac ggcatatctg 300 cagatcagca gcctaaaggc tgaggacact gccatatatt actgtgcgcc ccgatatagc 360 agcagctggt accttgatta ctggggccag ggaaccctgg tcaccgtgtc ctcagctagc 420 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 480 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 540 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 660 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga gcccaaatct 720 Page 12 961908Seq.TXT tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtatgtg 900 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 960 taccgtgtgg tcagcgtcct caccgtcctg caccaagact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1140 aagaaccaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 tccgacggct ccttcttcct ctattccaag ctcaccgtgg acaagagcag gtggcagcag 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1380 agcctctccc tgtctccggg caaatga 1407 <210> 23 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 23 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattcagag 60 gttcagctgc agcagtctgg ggctgagctt gtgaggccag gggccttggt caagttgtcc 120 tgcaaaactt ctggcttcaa cattaaagac tactttttac actgggtgag acagaggcct 180 gaccagggcc tggagtggat tggatggatt aatcctgata atggtaatac tgtttatgac 240 ccgaagtttc agggcacggc cagtttaaca gcagacacat cctccaacac agtctacttg 300 cagctcagcg gcctgacatc tgaggacact gccgtctatt tctgtactcg gagggactat 360 acttatgaaa aggctgctct ggactactgg ggtcagggag cctcagtcat cgtctcctca 420 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 720 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900 tatgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aagactggct gaatggcaag 1020 Page 13 961908Seq.TXT gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1140 atgaccaaga accaagtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260 ctggactccg acggctcctt cttcctctat tccaagctca ccgtggacaa gagcaggtgg 1320 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380 cagaagagcc tctccctgtc tccgggcaaa tga 1413 <210> 24 <211> 234 <212> PRT <213> Homo sapiens <400> 24 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val 35 40 45 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn 100 105 110 Trp Leu Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Page 14 961908Seq.TXT 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 25 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 25 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ala Ile Gln Met Ser Gln Ser Pro Ala Ser Leu Ser Ala 20 25 30 Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile 35 40 45 Tyr Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro His 50 55 60 Leu Leu Val Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Ser 85 90 95 Leu Gln Thr Glu Asp Ser Gly Asn Tyr Tyr Cys Gln His Phe Trp Ser 100 105 110 Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Page 15 961908Seq.TXT Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 26 <211> 705 <212> DNA <213> Homo sapiens <400> 26 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattcagaa 60 attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 120 tcctgcaggg ccagtcagag tgttagcagc tacttagcct ggtaccaaca gaaacctggc 180 caggctccca ggctcctcat ctatgatgca tccaacaggg ccactggcat cccagccagg 240 ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 300 gattttgcag tttattactg tcagcagcgt agcaactggc tcatgtacac ttttggccag 360 gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 705 <210> 27 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattcagcc 60 atccagatgt ctcagtctcc agcctcccta tctgcatctg tgggagaaac tgtcaccatc 120 acatgtcgag caagtggaaa tatttacaat tatttagcat ggtatcagca gaaacaggga 180 aaatctcctc acctcctggt ctatgatgca aaaaccttag cagatggtgt gccatcaagg 240 ttcagtggca gtggctcagg gacacaatat tctctcaaga ttagcagcct gcagactgaa 300 gattctggga attattactg tcaacatttt tggagtcttc cattcacgtt cggctcgggg 360 Page 16 961908Seq.TXT acaaagttgg aaataaaacg aactgtggct gcaccatctg tcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ag 702 <210> 28 <211> 537 <212> PRT <213> Homo sapiens <400> 28 Met Ser Ala Pro Lys Leu Leu Ser Leu Gly Cys Ile Phe Phe Pro Leu 1 5 10 15 Leu Leu Phe Gln Gln Ala Arg Ala Gln Phe Pro Arg Gln Cys Ala Thr 20 25 30 Val Glu Ala Leu Arg Ser Gly Met Cys Cys Pro Asp Leu Ser Pro Val 35 40 45 Ser Gly Pro Gly Thr Asp Arg Cys Gly Ser Ser Ser Gly Arg Gly Arg 50 55 60 Cys Glu Ala Val Thr Ala Asp Ser Arg Pro His Ser Pro Gln Tyr Pro 65 70 75 80 His Asp Gly Arg Asp Asp Arg Glu Val Trp Pro Leu Arg Phe Phe Asn 85 90 95 Arg Thr Cys His Cys Asn Gly Asn Phe Ser Gly His Asn Cys Gly Thr 100 105 110 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asp Gln Arg Val Leu Ile 115 120 125 Val Arg Arg Asn Leu Leu Asp Leu Ser Lys Glu Glu Lys Asn His Phe 130 135 140 Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Leu Phe Val 145 150 155 160 Ile Ala Thr Arg Arg Ser Glu Glu Ile Leu Gly Pro Asp Gly Asn Thr 165 170 175 Pro Gln Phe Glu Asn Ile Ser Ile Tyr Asn Tyr Phe Val Trp Thr His 180 185 190 Page 17 961908Seq.TXT Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Val Gly Gln Glu Ser Phe 195 200 205 Gly Glu Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His 210 215 220 Arg Tyr His Leu Leu Arg Leu Glu Lys Asp Met Gln Glu Met Leu Gln 225 230 235 240 Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn 245 250 255 Val Cys Asp Ile Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe 260 265 270 Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val 275 280 285 Val Cys Asp Ser Leu Glu Asp Tyr Asp Thr Leu Gly Thr Leu Cys Asn 290 295 300 Ser Thr Glu Asp Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Ala 305 310 315 320 Arg Pro Met Val Gln Arg Leu Pro Glu Pro Gln Asp Val Ala Gln Cys 325 330 335 Leu Glu Val Gly Leu Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr 340 345 350 Asn Ser Phe Arg Asn Thr Val Glu Gly Tyr Ser Asp Pro Thr Gly Lys 355 360 365 Tyr Asp Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu 370 375 380 Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Asp Pro Ile Phe 385 390 395 400 Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg 405 410 415 Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile 420 425 430 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr 435 440 445 Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Thr Tyr 450 455 460 Page 18 961908Seq.TXT Glu Ile Gln Trp Pro Ser Arg Glu Phe Ser Val Pro Glu Ile Ile Ala 465 470 475 480 Ile Ala Val Val Gly Ala Leu Leu Leu Val Ala Leu Ile Phe Gly Thr 485 490 495 Ala Ser Tyr Leu Ile Arg Ala Arg Arg Ser Met Asp Glu Ala Asn Gln 500 505 510 Pro Leu Leu Thr Asp Gln Tyr Gln Cys Tyr Ala Glu Glu Tyr Glu Lys 515 520 525 Leu Gln Asn Pro Asn Gln Ser Val Val 530 535 <210> 29 <211> 449 <212> PRT <213> Homo sapiens <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Cys Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Page 19 961908Seq.TXT Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Page 20 961908Seq.TXT Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 30 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Ser Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Page 21 961908Seq.TXT Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 31 <211> 452 Page 22 961908Seq.TXT <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 31 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Leu Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Phe Leu His Trp Val Arg Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Thr Ala Ser Leu Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Ser Gly Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Ala Ser Val Ile Val Phe Ser Ala Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Page 23 961908Seq.TXT Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 32 <211> 215 <212> PRT <213> Homo sapiens <400> 32 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Page 24 961908Seq.TXT 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Leu Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 33 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 33 Ala Ile Gln Met Ser Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr 20 25 30 Page 25 961908Seq.TXT Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro His Leu Leu Val 35 40 45 Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Ser Leu Gln Thr 65 70 75 80 Glu Asp Ser Gly Asn Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 Page 26